| Assay Method Information | |
| | Biochemical Assay |
| Description: | The kinase assay was carried out in streptavidin-coated 348-well microtitre FlashPlates . To this end, 1.5 μg of the DNA-PK/protein complex and 100 ng of biotinylated substrate, such as, for example, PESQEAFADLWKK biotin-NH2 (biotin-DNA-PK peptide), in a total volume of 36.5 μl (34.25 mM HEPES/KOH, 7.85 mM Tris-HCl, 68.5 mM KCl, 5 μM ATP, 6.85 mM MgCl2, 0.5 mM EDTA, 0.14 mM EGTA, 0.69 mM DTT, pH 7.4), were incubated at room temperature for 90 min with 500 ng of DNA from calf thymus, 0.1 μCi of 33P-ATP and 1.8% of DMSO per well with or without the test compound. The reaction was stopped using 50 μl/well of 200 mM EDTA. After incubation for a further 30 min at room temperature, the liquid was removed. Each well was washed three times with 100 μl of 0.9% sodium chloride solution. A non-specific reaction (blank value) was determined using 10 μM of a proprietary kinase inhibitor. |
| Affinity data for this assay | |
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